Difference between revisions of "Team:Nanjing-China/Notebook"

♠ We established Nanjing-China 2017 in March this year. Both the team leader and all team members are new iGEMers, in which case great challenges were ahead of us. We determined that we work on gas sensors and then read a lot of papers and conceived the outline of our project.

♥ In April, we started synthesizing primers and genes with the help of Sangon BiotechÒ and GenscriptÒ. We also made a proposal within School of Life Science to secure funding and support.

♣ From May to August, we were busy with lab work and optimizing experiments. At the end of August, we sent 3 students to attend CCIC in Fuzhou, Fujian and asked iGEMers from all over China to fill in our questionnaires so that we got to know what they thought about our project.

♦ In September and October, we visited AddiseoÒ in Nanjing to learn about biosafety issues, did all the other human practices and formed collaboration with team OUC-China. We also spread no effort in building wiki and modeling before November. We had been making beautiful original design throughout the whole time. We were so lucky to have talented and hard-working students to get everything neatly done before the Giant Jamboree.

DNA purification/Axygen gel extraction

1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination.
2. Absorb the liquids left on the surface of the gel slices using paper towels. Weigh gel slice (tare with empty tube).
3. Add 3 volumes of DE-A buffer per mg of gel (so a 100mg gel gets 300ul of buffer).
4. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved (keep heating for 6-8 minutes). If low-melt agarose gel is used, you may heat it at 40℃. Intermittently vortexing every 2-3 minutes will do a lot of help to accelerate the solubilization.
Note: Buffer DE-A is red liquid, so you can observe the color to make sure the gel is fully dissolved.
5. Add 0.5× Buffer DE-A volume of Buffer DE-B and mix. If the DNA fragment is less than 400bp, supplement further with a 1×sample volume of isopropanol.
Note: After the addition of DE-B, the solution should be in the uniform color of yellow.
6. Place a Miniprep column into a 2ml microfuge tube (provided). Transfer the solubilized agarose from the step above into the column. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7. Return the Miniprep column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
8. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
9．Place the Miniprep column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
10. Place the Miniprep column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
 11. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 50ul of ddH2O to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
 Note: Pre-warm the ddH₂O at 65℃ will generally improve elution efficiency.

Preparation of chemically competent E.coli cells

1. Inoculate 2ml LB broth with an aliquot (about 50ul)of the desired E.coli from the -80℃ freezer stock of cells.
2. Incubate for 2h at 37℃.
3. Add the 2ml seed culture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).
4. Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl^2-glycerine (0.1mol/L CaCl₂) and CaCl^2- MgCl₂ (80mmol/L MgCl₂, 20mmol/L CaCl₂). Set the centrifuge and prepare the ice tray.
5. Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes.
6. Centrifuge at 4℃, 4100rpm for 10 minutes.
7. Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out.
8. Add 30ml of pre-cooled CaCl^2- MgCl₂ per 50ml of initial liquid medium to resuspend bacteria cell pellet.
9. Centrifuge at 4℃, 4100rpm for 10 minutes.
10. Discard supernatant then place the tube upside down to make sure trace liquid medium runs out.
11. Add 2ml of pre-cooled CaCl₂ per 50ml of initial liquid medium to resuspend bacteria cell pellet.
12. Transfer to EP tubes (50ul every tube) and store at -80℃.

General Heat-Shock Transformation

1. Add 10ul DNA to 50ul cells on ice (set negative control by using chemically competent E.coli cells without plasmids).
2. Incubate on ice for 30 minutes.
3. Heat shock at 42℃ for exactly 90 seconds.
4. Place samples back on ice for 1-2 minutes.
5. Operating in the clean bench, add 900ul of LB broth per tube.
6. Incubate at 37℃ for 60 minutes, shaking.
7. Activate it on the plate for 60 minutes. The total number of plates is 7.
8. Centrifuge at 3000rpm for 1 minute.
9. Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.
10. Use the inoculating loop to load bacteria liquid then streak on the LB plate.
11. Place plates upside down and incubate at 37℃ overnight.

PCR method/Taq PCR

1. Thaw Taq, dNTP, primers, template DNA on ice.
2. To a new PCR tube, add:

template DNA	1ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq/La Taq E	1ul
ddH2O	37ul
total	50ul

3. Mix solution well.
4. Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 3min at 95℃;
Loop (29 cycles),
Denaturation: 30s at 95℃,
Annealing: 1min per 1k bp at 60℃,
Elongation: 1min at 72℃;
Final elongation: 10min at 72℃;
Store: 12℃.(not for too long).
5. We use 5ul of the PCR product for electrophoresis and 45ul for purification (details see DNA purification/AxyPrep PCR DNA purification PCR).

DNA purification/AxyPrep PCR DNA purification PCR

1. Add 3 volumes of Buffer PCR-A to the solution (if Buffer PCR-A is less than 100ul, then add to 100 ul). Mix gently and then transfer to a Miniprep column, which is placed in a 2ml microfuge tube (provided).
2. Centrifuge at 12,000rpm for 1 minute and discard the filtrate from the 2ml microfuge tube.
3. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
4. Return the Miniprep column to the 2ml microfuge tube and add 400ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Note: this step can be omitted.
5. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 25-30ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

Plasmid extraction

1. Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.
2. Add 250ul of Buffer S1 to the pellet to resuspend bacteria cells.
3. Add 250ul of Buffer S2, mix gently by inverting the tube 4-6 times until the solution becomes clear. The time should be no longer than 5 minutes.
4. Add 350ul of Buffer S3, mix gently by inverting the tube 6-8 times.
5. Centrifuge at 12,000rpm for 10 minutes.
6. Place spin column into a 2ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7. Return the column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
8. Return the column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
9. Place the column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
10. Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
11. Transfer the column into a clean 1.5ml microfuge tube (provided). Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

Agarose Gel Electrophoresis

1. Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).
2. Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).
3. Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb.
4. Let the gel cool until it becomes solid.
5. Pull out the comb carefully.
6. Place the gel in the electrophoresis chamber.
7. Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.
8. Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.
9. Run the gel at 135V for about twenty minutes.

Colony PCR

1. We carry out colony PCR in order to amplify a few copies of DNA across several orders of magnitude and check the length of DNA sequences between two designed primers.
2. To 20 new PCR tubes (add 2ul bacteria cells and operate thermal cracking at 95℃ for 15 minutes), add:

template DNA	2ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq/La Taq E	1ul
ddH2O	36ul
total	50ul

3. To 2 new PCR tubes , add:

template DNA	1ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq/La Taq E	1ul
ddH2O	37ul
total	50ul

4. Mix solution well.
5. Place tube in PCR thermocycler. Set thermocycler program:
Loop (29 cycles),
Denaturation: 30s at 95℃,
Annealing: 1min per 1k bp at 60℃,
Elongation: 1min at 72℃;
Final elongation: 10min at 72℃;
Store: 12℃.(not for too long).
6. use the PCR product for electrophoresis.

Protocol for restriction enzyme digestion

To a 1.5ml microfuge tube, add

Insert	17ul
EcoRⅠ	1ul
PstⅠ	1ul
10×K	1ul
total	20ul

Protocol for DNA ligation

1. To a 1.5ml microfuge tube, add:

Vector	1ul
Insert	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

2. To a 1.5ml microfuge tube (control), add: 

Vector	1ul
ddH2O	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

3. To a 1.5ml microfuge tube, add:

Vector	1ul
Insert	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

4. Incubate at 16℃. 

Protein Inducing

A. hydrogen
B. formaldehyde
1. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium using streak plate method.
2. After culturing on LB-agar at 37℃ for 12 hours, pick a single colony, shake in 10mL LB liquid buffer, 37℃, 160rpm, for 8 hours over night.
3. Extract 1mL from 10mL sanitized LB liquid buffer, measure its OD600 (Eppendorf TM Biophotometer plus) as blank. Add the 10mL product of step ii into 200mL LB liquid buffer.
4. Shake for about 2 hours, 37℃, 160rpm, until OD600 reaches ~0.5
5. Divide the product of step iv into eleven serum bottles, 20ml per bottle.
6. Use 500uM, 1000 uM, 8000 uM as the highest concentration to test the maximum tolerance, every 50uM or 100uM as the interval. Add different concentration of formaldehyde solution into the bottle as the table shows.

The concentration of formaldehyde

	1	2	3	4	5	6	7	8	9	10	11
①/uM	0	50	100	150	200	250	300	350	400	450	500
②/uM	0	100	200	300	400	500	600	700	800	900	1000
③/uM	0	800	2000	4000	5000	6000	7000	7250	7500	7750	8000

7. Shake at the same condition for 13h, inducing the expression of RFP protein.

8. Measure OD600 and fluorescence intensity with a microplate reader, and then draw the growth curve and the curve reflecting the relationship between the fluorescence intensity and the concentration.

C. hydrogen sulfide
1. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium for 12 hours, using streak plate method.

2. Pick a single colony and incubate it in 20mL Luria–Bertani liquid medium at 37 °C, shaking over night at 180rpm/min. Ampicillin was used at concentrations of 50μg/mL.
3. Add the bacteria solution into 200mL LB liquid medium and incubate at 37 °C, 180rpm/min for reactivation until OD600 reaches 0.5.
4. Prepare Na₂S solution:

	①	②	③	④	⑤	⑥	⑦	⑧	⑨	⑩
Ⅰ[c]/uM	0	7.8	15.6	31.25	62.5	123	250	500	1000	2000
Ⅱ[c]/uM	10	20	30	40	50	60	70	80	90	100

5. Divide the bacteria solution into 50mL centrifuge tubes, 15mL each. Add Na₂S solution to each tube at the designed concentrations.
6. Incubate for 17 hours at 37 °C, 180rpm/min.
7. Take 200μL bacteria solution from each tube. Measure OD600 and fluorescence intensity using a microplate reader after shaking for 10 seconds, with absorption wavelength at 587nm, emission wavelength at 610nm, and bandwidth at 8nm. Draw the fluorescence intensity-S2- concentration curve.

Test of Selectivity

A. hydrogen
B. formaldehyde
1. Prepare formaldehyde, acetaldehyde, propanone, DMSO, glyoxal, tribromoacetaldehyde, aldehyde aldehyde, glutaraldehyde sterile solution.

2. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium using streak plate method.

3. After culturing on LB-agar at 37℃ for 12 hours, pick a single colony, shake in 100mL LB liquid buffer, 37℃, 160rpm, for 8 hours over night.

4. Extract 1mL from 100mL sanitized LB liquid buffer, measure its OD600 (Eppendorf TM Biophotometer plus) as blank. Add the 10mL product of step ii into 200mL LB liquid buffer.

5. Shake for about 2 hours, 37℃, 160rpm, until OD600 reaches ~0.5 (OD600 should be measured after 1.5 hours and could be predicted using the formula OD600t+20min=2*OD600t).

6. Divide the product of step iv into eleven serum bottles, 20ml per bottle.

7. Add formaldehyde, acetaldehyde, propanone, DMSO, glyoxal, tribromoacetaldehyde, aldehyde aldehyde, glutaraldehyde sterile solution into the bottle, ensuring that the final concentration of the solution is 800uM.

8. Shake at the same condition for 13h, inducing the expression of RFP protein.

9. Measure the fluorescence intensity by a microplate reader, and then draw the histogram to reflect the relationship between the fluorescence intensity and the type of concentration.

C. hydrogen sulfide
1. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium for 12 hours, using streak plate method.
2. Pick a single colony and incubate it in 20mL Luria–Bertani liquid medium at 37 °C, shaking over night at 180rpm/min. Ampicillin was used at concentrations of 50μg/mL.
3. Add the bacteria solution into 200mL LB liquid medium and incubate at 37 °C, 180rpm/min for reactivation until OD600 reaches 0.5.
4. Prepare solution:

inducer	S2-	SO42-	SO32-	S2O32-	SCN-	NAC	PO43-
[c]/μM	100

5. Divide the bacteria solution into 50mL centrifuge tubes, 15mL each. Add the solution to each tube.

6. Incubate for 17 hours at 37 °C, 180rpm/min

Protein inducing

Test of Selectivity